PML-like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication-based single-strand breaks.

DNA lesions induce recruitment and accumulation of various repair factors, resulting in formation of discrete nuclear foci. Using superresolution fluorescence microscopy as well as live cell and quantitative imaging, we demonstrate that X-ray repair cross-complementing protein 1 (XRCC1), a key factor in single-strand break and base excision repair, is recruited into nuclear bodies formed in response to replication-related single-strand breaks. Intriguingly, these bodies are assembled immediately in the vicinity of these breaks and never fully colocalize with replication foci. They are structurally organized, containing canonical promyelocytic leukemia (PML) nuclear body protein SP100 concentrated in a peripheral layer, and XRCC1 in the center. They also contain other factors, including PML, poly(ADP-ribose) polymerase 1 (PARP1), ligase IIIα, and origin recognition complex subunit 5. The breast cancer 1 and -2 C terminus domains of XRCC1 are essential for formation of these repair foci. These results reveal that XRCC1-contaning foci constitute newly recognized PML-like nuclear bodies that accrete and locally deliver essential factors for repair of single-strand DNA breaks in replication regions.-Kordon, M. M., Szczurek, A., Berniak, K., Szelest, O., Solarczyk, K., Tworzydlo, M., Wachsmann-Hogiu, S., Vaahtokari, A., Cremer, C., Pederson, T., Dobrucki, J. W. PML-like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication-based single-strand breaks.

Are plasmonic optical biosensors ready for use in point-of-need applications?

Plasmonics has drawn significant attention in the area of biosensors for decades due to the unique optical properties of plasmonic resonant nanostructures. While the sensitivity and specificity of molecular detection relies significantly on the resonance conditions, significant attention has been dedicated to the design, fabrication, and optimization of plasmonic substrates. The adequate choice of materials, structures, and functionality goes hand in hand with a fundamental understanding of plasmonics to enable the development of practical biosensors that can be deployed in real life situations. Here we provide a brief review of plasmonic biosensors detailing most recent developments and applications. Besides metals, novel plasmonic materials such as graphene are highlighted. Sensors based on Surface Plasmon Resonance (SPR), Localized Surface Plasmon Resonance (LSPR), and Surface Enhanced Raman Spectroscopy (SERS) are presented and classified based on their materials and structure. In addition, most recent applications to environment monitoring, health diagnosis, and food safety are presented. Potential problems related to the implementation in such applications are discussed and an outlook is presented.

A Bifunctional Anti-Amyloid Blocks Oxidative Stress and the Accumulation of Intraneuronal Amyloid-Beta.

There is growing recognition regarding the role of intracellular amyloid beta (Aß) in the Alzheimer's disease process, which has been linked with aberrant signaling and the disruption of protein degradation mechanisms. Most notably, intraneuronal Aß likely underlies the oxidative stress and mitochondrial dysfunction that have been identified as key elements of disease progression. In this study, we employed fluorescence imaging to explore the ability of a bifunctional small molecule to reduce aggregates of intracellular Aß and attenuate oxidative stress. Structurally, this small molecule is comprised of a nitroxide spin label linked to an amyloidophilic fluorene and is known as spin-labeled fluorene (SLF). The effect of the SLF on intracellular Aß accumulation and oxidative stress was measured in MC65 cells, a human neuronal cell line with inducible expression of the amyloid precursor protein and in the N2a neuronal cell line treated with exogenous Aß. Super-resolution microscopy imaging showed SLF decreases the accumulation of intracellular Aß. Confocal microscopy imaging of MC65 cells treated with a reactive oxygen species (ROS)-sensitive dye demonstrated SLF significantly reduces the intracellular Aß-induced ROS signal. In order to determine the contributions of the separate SLF moieties to these protective activities, experiments were also carried out on cells with nitroxides lacking the Aß targeting domain or fluorene derivatives lacking the nitroxide functionality. The findings support a synergistic effect of SLF in counteracting both the conformational toxicity of both endogenous and exogenous Aß, its promotion of ROS, and Aß metabolism. Furthermore, these studies demonstrate an intimate link between ROS production and Aß oligomer formation.

Fluorescence-suppressed time-resolved Raman spectroscopy of pharmaceuticals using complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector.

In this work, we utilize a short-wavelength, 532-nm picosecond pulsed laser coupled with a time-gated complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector to acquire Raman spectra of several drugs of interest. With this approach, we are able to reveal previously unseen Raman features and suppress the fluorescence background of these drugs. Compared to traditional Raman setups, the present time-resolved technique has two major improvements. First, it is possible to overcome the strong fluorescence background that usually interferes with the much weaker Raman spectra. Second, using the high photon energy excitation light source, we are able to generate a stronger Raman signal compared to traditional instruments. In addition, observations in the time domain can be performed, thus enabling new capabilities in the field of Raman and fluorescence spectroscopy. With this system, we demonstrate for the first time the possibility of recording fluorescence-suppressed Raman spectra of solid, amorphous and crystalline, and non-photoluminescent and photoluminescent drugs such as caffeine, ranitidine hydrochloride, and indomethacin (amorphous and crystalline forms). The raw data acquired by utilizing only the picosecond pulsed laser and a CMOS SPAD detector could be used for identifying the compounds directly without any data processing. Moreover, to validate the accuracy of this time-resolved technique, we present density functional theory (DFT) calculations for a widely used gastric acid inhibitor, ranitidine hydrochloride. The obtained time-resolved Raman peaks were identified based on the calculations and existing literature. Raman spectra using non-time-resolved setups with continuous-wave 785- and 532-nm excitation lasers were used as reference data. Overall, this demonstration of time-resolved Raman and fluorescence measurements with a CMOS SPAD detector shows promise in diverse areas, including fundamental chemical research, the pharmaceutical setting, process analytical technology (PAT), and the life sciences.

Smart and Fast Blood Counting of Trace Volumes of Body Fluids from Various Mammalian Species Using a Compact, Custom-Built Microscope Cytometer.

We report an accurate method to count red blood cells, platelets, and white blood cells, as well as to determine hemoglobin in the blood of humans, horses, dogs, cats, and cows. Red and white blood cell counts can also be performed on human body fluids such as cerebrospinal fluid, synovial fluid, and peritoneal fluid. The approach consists of using a compact, custom-built microscope to record large field-of-view, bright-field, and fluorescence images of samples that are stained with a single dye and using automatic algorithms to count blood cells and detect hemoglobin. The total process takes about 15 min, including 5 min for sample preparation, and 10 min for data collection and analysis. The minimum volume of blood needed for the test is 0.5 µL, which allows for minimally invasive sample collection such as using a finger prick rather than a venous draw. Blood counts were compared to gold-standard automated clinical instruments, with excellent agreement between the two methods as determined by a Bland-Altman analysis. Accuracy of counts on body fluids was consistent with hand counting by a trained clinical lab scientist, where our instrument demonstrated an approximately 100-fold lower limit of detection compared to current automated methods. The combination of a compact, custom-built instrument, simple sample collection and preparation, and automated analysis demonstrates that this approach could benefit global health through use in low-resource settings where central hematology laboratories are not accessible.

Combined fiber probe for fluorescence lifetime and Raman spectroscopy.

In this contribution we present a dual modality fiber optic probe combining fluorescence lifetime imaging (FLIm) and Raman spectroscopy for in vivo endoscopic applications. The presented multi-spectroscopy probe enables efficient excitation and collection of fluorescence lifetime signals for FLIm in the UV/visible wavelength region, as well as of Raman spectra in the near-IR for simultaneous Raman/FLIm imaging. The probe was characterized in terms of its lateral resolution and distance dependency of the Raman and FLIm signals. In addition, the feasibility of the probe for in vivo FLIm and Raman spectral characterization of tissue was demonstrated. Graphical Abstract An image comparison between FLIm and Raman spectroscopy acquired with the bimodal probe onseveral tissue samples.

Image reconstruction for structured-illumination microscopy with low signal level.

We report a new image processing technique for the structured illumination microscopy designed to work with low signals, with the goal of reducing photobleaching and phototoxicity of the sample. Using a pre-filtering process to estimate experimental parameters and total variation as a constraint to reconstruct, we obtain two orders of magnitude of exposure reduction while maintaining the resolution improvement and image quality compared to a standard structured illumination microscopy. The algorithm is validated on both fixed and live cell data with results confirming that we can image more than 15x more time points compared to the standard technique.

Optimization and miniaturization of SE-ECL for potential-resolved, multi-color, multi-analyte detection.

Electrochemiluminescence (ECL) is a bioanalytical technique with numerous advantages, including the potential for high temporal and spatial resolution, a high signal-to-noise ratio, a broad dynamic range, and rapid measurement capabilities. To reduce the complexity of a multi-electrode approach, we use a single-electrode electrochemiluminescence (SE-ECL) configuration to achieve the simultaneous emission and detection of multiple colors for applications that require multiplexed detection of several analytes. This method exploits intrinsic differences in the electric potential applied along single electrodes built into electrochemical cells, enabling the achievement of distinct colors through selective excitation of ECL luminophores. We present results on the optimization of SE-ECL intensity for different channel lengths and widths, with sum intensities being 5 times larger for 6 cm vs. 2 cm channels and linearly increasing with the width of the channels. Furthermore, we demonstrated for the first time that applying Alternating Current (AC) voltage within the single electrode setup for driving the ECL reactions has a dramatic effect on the emitted light intensity, with square waveforms resulting in higher intensities vs sine waveforms. Additionally, multiplexed multicolor SE-ECL on a 6.5 mm × 3.6 mm CMOS semiconductor image sensor was demonstrated for the first time, with the ability to simultaneously distinguish four different colors, leading to the ability to measure multiple analytes.

On-chip bioluminescence biosensor for the detection of microbial surface contamination.

Detection of microbial pathogens is important for food safety reasons, and for monitoring sanitation in laboratory environments and health care settings. Traditional detection methods such as culture-based and nucleic acid-based methods are time-consuming, laborious, and require expensive laboratory equipment. Recently, ATP-based bioluminescence methods were developed to assess surface contamination, with commercial products available. In this study, we introduce a biosensor based on a CMOS image sensor for ATP-mediated chemiluminescence detection. The original lens and IR filter were removed from the CMOS sensor revealing a 12 MP periodic microlens/pixel array on an area of 6.5 mm × 3.6 mm. UltraSnap swabs are used to collect samples from solid surfaces including personal electronic devices, and office and laboratory equipment. Samples mixed with chemiluminescence reagents were placed directly on the surface of the image sensor. Close proximity of the sample to the photodiode array leads to high photon collection efficiency. The population of microorganisms can be assessed and quantified by analyzing the intensity of measured chemiluminescence. We report a linear range and limit of detection for measuring ATP in UltraSnap buffer of 10-1000 nM and 225 fmol, respectively. The performance of the CMOS-based device was compared to a commercial luminometer, and a high correlation with a Pearson's correlation coefficient of 0.98589 was obtained. The Bland-Altman plot showed no significant bias between the results of the two methods. Finally, microbial contamination of different surfaces was analyzed with both methods, and the CMOS biosensor exhibited the same trend as the commercial luminometer.

Diatomite-Based, Flexible SERS Immunosensor Platform for Rapid, Specific, and Sensitive Detection of Circulating Cancer-Specific Protein Biomarkers in Serum Using Raman Probes.

Cancer is one of the most actively researched diseases having a high mortality rate when not detected at an early stage. Thus, rapid, simultaneous, and sensitive quantification of cancer biomarkers plays an important role in early diagnosis, with patient impact to disability adjusted life years. Herein, a diatomite-based SERS flexible platform for the rapid and sensitive detection of circulating cancer-specific protein biomarkers in serum is presented. In this approach, diatomite/AgNPs strips with maximum SERS activity prepared using the layer-by-layer (LbL) technique were modified with specific antibodies, and specific antigens (HER2, CA15-3, PSA, and MUC4) were captured and detected. By using Raman probes specific to the captured antigens in serum, a SERS limit of detection (LOD) of 0.1 ng/mL was measured (calculated LOD < 0.1 ng/mL). This value is lower than the cutoff amount of cancer antigens in the person's blood. The specificity for the antigens of each antibody was calculated to be higher than 95%. As a result, an immunosensor for rapid detection of cancer biomarkers in serum with good specificity, high sensitivity, good reproducibility, and low cost has been demonstrated. Overall, we show that the prepared diatomite-based SERS substrate with a high surface-to-volume ratio is a useable platform for immunoassay tests.

Hydrophobicity-driven self-assembly of protein and silver nanoparticles for protein detection using surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) is a promising analytical technique for the detection and characterization of biological molecules and structures. The role of hydrophobic and hydrophilic surfaces in the self-assembly of protein-metallic nanoparticle structures for label-free protein detection is demonstrated. Aggregation is driven by both the hydrophobicity of the surface as well as the charge of the proteins. The best conditions for obtaining a reproducible SERS signal that allows for sensitive, label-free protein detection are provided by the use of hydrophobic surfaces and 16 × 10(11) NPs per mL. A detection limit of approximately 0.5 µg mL(-1) is achieved regardless of the proteins' charge properties and size. The developed method is simple and can be used for reproducible and sensitive detection and characterization of a wide variety of biological molecules and various structures with different sizes and charge status.

Mutations in the gene encoding filamin B disrupt vertebral segmentation, joint formation and skeletogenesis.

The filamins are cytoplasmic proteins that regulate the structure and activity of the cytoskeleton by cross-linking actin into three-dimensional networks, linking the cell membrane to the cytoskeleton and serving as scaffolds on which intracellular signaling and protein trafficking pathways are organized (reviewed in refs. 1,2). We identified mutations in the gene encoding filamin B in four human skeletal disorders. We found homozygosity or compound heterozygosity with respect to stop-codon mutations in autosomal recessive spondylocarpotarsal syndrome (SCT, OMIM 272460) and missense mutations in individuals with autosomal dominant Larsen syndrome (OMIM 150250) and the perinatal lethal atelosteogenesis I and III phenotypes (AOI, OMIM 108720; AOIII, OMIM 108721). We found that filamin B is expressed in human growth plate chondrocytes and in the developing vertebral bodies in the mouse. These data indicate an unexpected role in vertebral segmentation, joint formation and endochondral ossification for this ubiquitously expressed cytoskeletal protein.

A deconvolution approach to modelling surges in COVID-19 cases and deaths.

The COVID-19 pandemic continues to emphasize the importance of epidemiological modelling in guiding timely and systematic responses to public health threats. Nonetheless, the predictive qualities of these models remain limited by their underlying assumptions of the factors and determinants shaping national and regional disease landscapes. Here, we introduce epidemiological feature detection, a novel latent variable mixture modelling approach to extracting and parameterizing distinct and localized features of real-world trends in daily COVID-19 cases and deaths. In this approach, we combine methods of peak deconvolution that are commonly used in spectroscopy with the susceptible-infected-recovered-deceased model of disease transmission. We analyze the second wave of the COVID-19 pandemic in Israel, Canada, and Germany and find that the lag time between reported cases and deaths, which we term case-death latency, is closely correlated with adjusted case fatality rates across these countries. Our findings illustrate the spatiotemporal variability of both these disease metrics within and between different disease landscapes. They also highlight the complex relationship between case-death latency, adjusted case fatality rate, and COVID-19 management across various degrees of decentralized governments and administrative structures, which provides a retrospective framework for responding to future pandemics and disease outbreaks.

MoS2-Plasmonic Nanocavities for Raman Spectra of Single Extracellular Vesicles Reveal Molecular Progression in Glioblastoma.

Extracellular vesicles (EVs) are continually released from cancer cells into biofluids, carrying actionable molecular fingerprints of the underlying disease with considerable diagnostic and therapeutic potential. The scarcity, heterogeneity and intrinsic complexity of tumor EVs present a major technological challenge in real-time monitoring of complex cancers such as glioblastoma (GBM). Surface-enhanced Raman spectroscopy (SERS) outputs a label-free spectroscopic fingerprint for EV molecular profiling. However, it has not been exploited to detect known biomarkers at the single EV level. We developed a multiplex fluidic device with embedded arrayed nanocavity microchips (MoSERS microchip) that achieves 97% confinement of single EVs in a minute amount of fluid (<10 µL) and enables molecular profiling of single EVs with SERS. The nanocavity arrays combine two featuring characteristics: (1) An embedded MoS2 monolayer that enables label-free isolation and nanoconfinement of single EVs due to physical interaction (Coulomb and van der Waals) between the MoS2 edge sites and the lipid bilayer; and (2) A layered plasmonic cavity that enables sufficient electromagnetic field enhancement inside the cavities to obtain a single EV level signal resolution for stratifying the molecular alterations. We used the GBM paradigm to demonstrate the diagnostic potential of the SERS single EV molecular profiling approach. The MoSERS multiplexing fluidic achieves parallel signal acquisition of glioma molecular variants (EGFRvIII oncogenic mutation and MGMT expression) in GBM cells. The detection limit of 1.23% was found for stratifying these key molecular variants in the wild-type population. When interfaced with a convolutional neural network (CNN), MoSERS improved diagnostic accuracy (87%) with which GBM mutations were detected in 12 patient blood samples, on par with clinical pathology tests. Thus, MoSERS demonstrates the potential for molecular stratification of cancer patients using circulating EVs.

Super-resolved spatial light interference microscopy.

We report a scheme to achieve resolution beyond the diffraction limit in spatial light interference microscopy (SLIM). By adding a grating to the optical path, the structured illumination technique can be used to improve the resolution by a factor of 2. We show that a direct application of the structured illumination technique, however, has proved to be unsuccessful. Through two crucial modifications, namely, one to the pupil plane of the objective and the other to the demodulation procedure, faithful phase information of the object is recovered and the resolution is improved by a factor of 2.

Characterization of high-affinity peptides and their feasibility for use in nanotherapeutics targeting leukemia stem cells.

Peptides featuring the LR(S/T) motif were identified that could specifically bind to the C-type lectin-like molecule-1 (CLL1), a protein preferentially expressed on acute myeloid leukemia stem cells (LSCs). Micellar nanoparticles were covalently decorated with CLL1-targeting peptides for targeted drug delivery. The resulting peptide-coated nanoparticles were 13.5 nm in diameter and could be loaded with 5 mg of daunorubicin per 20 mg of telodendrimers. These "targeting nanomicelles" transported the drug load to the interior of cells expressing CLL1 and to LSCs isolated from clinical specimens in vitro, but did not bind to normal blood or normal hematopoietic stem cells. The presence of CLL1-targeting peptides on the surface of the nanomicelles enabled the improved binding and delivery of substantially more daunorubicin into the cells expressing CLL1 and CD34(+) leukemic cells compared with unmodified nanomicelles. In conclusion, nanomicelles coated with CLL1-targeting peptides are potentially useful for eradicating LSCs and improving leukemia therapy. FROM THE CLINICAL EDITOR: Micellar nanoparticles covalently decorated with targeting peptides were used for targeted drug delivery of daunorubicin to address acute myeloid leukemia stem cells.

Progress and Challenges of Point-of-Need Photonic Biosensors for the Diagnosis of COVID-19 Infections and Immunity.

The new coronavirus disease, COVID-19, caused by SARS-CoV-2, continues to affect the world and after more than two years of the pandemic, approximately half a billion people are reported to have been infected. Due to its high contagiousness, our life has changed dramatically, with consequences that remain to be seen. To prevent the transmission of the virus, it is crucial to diagnose COVID-19 accurately, such that the infected cases can be rapidly identified and managed. Currently, the gold standard of testing is polymerase chain reaction (PCR), which provides the highest accuracy. However, the reliance on centralized rapid testing modalities throughout the COVID-19 pandemic has made access to timely diagnosis inconsistent and inefficient. Recent advancements in photonic biosensors with respect to cost-effectiveness, analytical performance, and portability have shown the potential for such platforms to enable the delivery of preventative and diagnostic care beyond clinics and into point-of-need (PON) settings. Herein, we review photonic technologies that have become commercially relevant throughout the COVID-19 pandemic, as well as emerging research in the field of photonic biosensors, shedding light on prospective technologies for responding to future health outbreaks. Therefore, in this article, we provide a review of recent progress and challenges of photonic biosensors that are developed for the testing of COVID-19, consisting of their working fundamentals and implementation for COVID-19 testing in practice with emphasis on the challenges that are faced in different development stages towards commercialization. In addition, we also present the characteristics of a biosensor both from technical and clinical perspectives. We present an estimate of the impact of testing on disease burden (in terms of Disability-Adjusted Life Years (DALYs), Quality Adjusted Life Years (QALYs), and Quality-Adjusted Life Days (QALDs)) and how improvements in cost can lower the economic impact and lead to reduced or averted DALYs. While COVID19 is the main focus of these technologies, similar concepts and approaches can be used and developed for future outbreaks of other infectious diseases.

SE-ECL on CMOS: a miniaturized electrochemiluminescence biosensor.

Biosensors exhibit high potential for the detection of analytes of interest at the point-of-need. Over the past two decades, the combination of novel biosensing systems - such as electrochemiluminescence (ECL) biosensors - and advances in microfluidic techniques has allowed the development of lab-on-a-chip devices with enhanced overall performance and simplified sample handling. However, recording data with conventional platforms requires advanced and complicated instruments, such as sensitive photodetectors coupled to microscopes, to capture the photons from the chemiluminescent reaction. In this work, we integrated microfluidic and luminol/hydrogen peroxide ECL systems on a complementary metal-oxide-semiconductor (CMOS) chip for sample handling and data collection on the same platform. This was achieved by the adaptation of a single electrode as an electrochemical transducer and a CMOS chip as a built-in detector. We demonstrated the application of this platform for the detection of uric acid (UA), a biomarker of gout disease. A linear detection range was observed from 25 to 300 µM, with a detection limit (LOD) as low as 26.09 µM. The device showed high reusability and reproducibility within the linear detection range while maintaining high selectivity for UA detection. The analytical performance has also been evaluated in simulated saliva and urine samples, demonstrating the potential utility in medical diagnosis at the point-of-need. Compared to other ECL imaging platforms, this device showed an eightfold increase in photon collection efficiency. Overall, this approach has promising potential as an inexpensive, portable, and efficient ECL platform for measuring analytes at the point-of-need.

Recent advances in optical label-free characterization of extracellular vesicles.

Extracellular vesicles (EVs) are complex biological nanoparticles endogenously secreted by all eukaryotic cells. EVs carry a specific molecular cargo of proteins, lipids, and nucleic acids derived from cells of origin and play a significant role in the physiology and pathology of cells, organs, and organisms. Upon release, they may be found in different body fluids that can be easily accessed via noninvasive methodologies. Due to the unique information encoded in their molecular cargo, they may reflect the state of the parent cell and therefore EVs are recognized as a rich source of biomarkers for early diagnostics involving liquid biopsy. However, body fluids contain a mixture of EVs released by different types of healthy and diseased cells, making the detection of the EVs of interest very challenging. Recent research efforts have been focused on the detection and characterization of diagnostically relevant subpopulations of EVs, with emphasis on label-free methods that simplify sample preparation and are free of interfering signals. Therefore, in this paper, we review the recent progress of the label-free optical methods employed for the detection, counting, and morphological and chemical characterization of EVs. We will first briefly discuss the biology and functions of EVs, and then introduce different optical label-free techniques for rapid, precise, and nondestructive characterization of EVs such as nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy, surface plasmon resonance spectroscopy, Raman spectroscopy, and SERS spectroscopy. In the end, we will discuss their applications in the detection of neurodegenerative diseases and cancer and provide an outlook on the future impact and challenges of these technologies to the field of liquid biopsy via EVs.

Multivariate optical computing using a digital micromirror device for fluorescence and Raman spectroscopy.

A multivariate optical computer has been constructed consisting of a spectrograph, digital micromirror device, and photomultiplier tube that is capable of determining absolute concentrations of individual components of a multivariate spectral model. We present experimental results on ternary mixtures, showing accurate quantification of chemical concentrations based on integrated intensities of fluorescence and Raman spectra measured with a single point detector. We additionally show in simulation that point measurements based on principal component spectra retain the ability to classify cancerous from noncancerous T cells.